Multiple vitamin deficiencies additively increase the risk of incident fractures in Japanese postmenopausal women.

The associations of multiple vitamin deficiencies on incident fractures were uncertain, the relationships between serum vitamin markers and incident bone fractures were investigated in Japanese postmenopausal women. The number of deficiencies was additively associated with incident fracture after adjustment for possible confounding factors including the treatment of osteoporosis. INTRODUCTION: To evaluate the associations of multiple vitamin deficiencies on incident fractures, the relationships between serum vitamin markers and incident bone fractures were investigated in Japanese postmenopausal women. METHODS: This analysis used a subset of the ongoing cohort maintained by a primary care institution. Inclusion criteria of the present study were postmenopausal women aged ≥ 50 years, without vitamin supplementation and secondary osteoporosis. Baseline serum concentrations of 25-hydroxyvitamin D (25(OH)D), undercarboxylated osteocalcin (ucOC), and homocysteine (Hcy) were measured to assess vitamin D, vitamin K, and vitamin B, respectively. Since 25(OH) D positively relates to vitamin D, ucOC and Hcy negatively relate to vitamin K and vitamin B nutrients, respectively, the subjects with lower (25(OH)D) or higher (ucOC or Hcy) values than each median value was defined as subjects with the corresponding vitamin deficiency. Subjects were divided into four groups according to the number of deficiency: no deficiency, single deficiency, double deficiencies, and triple deficiencies. Relationships between the vitamin deficiencies and incident fractures were evaluated by Cox regression analysis. RESULTS: A total of 889 subjects were included in this analysis; their mean and SD age was 68.3 ± 9.5 years, and the follow-up period was 6.3 ± 5.1 years. The numbers of subjects in the four groups were 139 (15.6%), 304 (34.2%), 316 (35.5%), and 130 (14.6%) for the groups with no, single, double, and triple deficiencies, respectively. Incident fractures were observed in 264 subjects (29.7%) during the observation period. The number of deficiencies was significantly associated with incident fracture (hazard ratio 1.25, 95% confidence interval 1.04-1.50, P = 0.018) after adjustment for possible confounding factors including the treatment of osteoporosis. CONCLUSION: Accumulation of vitamin deficiencies was related to incident fractures.

Hyperthermic intraperitoneal chemotherapy using a combination of mitomycin C,5-fluorouracil, and oxaliplatin in patients at high risk of colorectal peritoneal metastasis: A Phase I clinical study.

INTRODUCTION: The drugs and protocols used for hyperthermic intraperitoneal chemotherapy (HIPEC) vary among institutions. Here we show the efficacy of the 3-drug combination of mitomycin C (MMC), 5-fluorouracil (5FU), and oxaliplatin (OHP) in an in vitro simulation of HIPEC and the safety of HIPEC with these drugs during a Phase I study of patients at high risk of developing colorectal peritoneal metastasis. METHODS: To simulate HIPEC, we used HCT116 and WiDr cells to assess the growth inhibitory efficacy of MMC 2 µg/mL, 5FU 200 µg/mL, and OHP 40 µg/mL as single drugs or their combination after an exposure time of 30 min at 37 or 42 °C. In addition, nine patients underwent surgical resection of tumors and HIPEC with MMC, 5FU, and an escalating dose of OHP (90/110/130 mg/m²). Dose-limiting toxicity was monitored. RESULTS: In the simulation, the 3-drug combination showed marked tumor-suppressive effects compared with those from ten times higher dose of OHP 400 µg/mL, with significant augmentation under hyperthermic conditions. No dose-limiting toxicity occurred in the clinical study. Dose escalation was completed at the final level of OHP. CONCLUSIONS: The MMC-5FU-OHP combination showed marked growth inhibition against colorectal cancer cells under hyperthermic conditions in vitro. In the phase I study, the recommended dose of OHP was determined as 130 mg/m² when used with MMC and 5FU; HIPEC using MMC-5FU-OHP appears to be safe and feasible for patients at high risk of colorectal peritoneal metastasis.

Effects of Rho-kinase inactivation on eosinophilia and hyper-reactivity in murine airways by allergen challenges.

BACKGROUND: A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. It is generally considered that eosinophilic inflammation and hyper-reactivity to methacholine in airways are fundamental to the pathophysiology of bronchial asthma. OBJECTIVE: This study was designed to determine whether the Rho/Rho-kinase pathways are involved in the eosinophil recruitment and airway hyper-reactivity. We investigated inhibitory effects of fasudil, a specific inhibitor of Rho-kinase, on acute allergic inflammation in mice. METHODS: BALB/c mice were sensitized and challenged with ovalbumin (OVA). OVA-challenged mice were treated orally with fasudil (3, 10, 30 mg/kg) or saline before each OVA challenge. Total cell counts, differential cell counts, cytokines, and chemokines levels were measured in bronchoalveolar lavage (BAL), and lungs were examined histologically. Moreover, respiratory resistance in response to methacholine was measured. RESULTS: When fasudil was administrated to OVA-challenged mice, increased cell numbers of total cells and eosinophils were significantly attenuated in a dose-dependent manner. However, inflammatory cells other than eosinophils were not affected by fasudil. Fasudil caused a dose-dependent inhibition in increased levels of IL-5, IL-13, and eotaxin in BAL fluid by OVA challenges. Histological analysis of the airways revealed that both infiltration of inflammatory cells and goblet cell hyperplasia were significantly suppressed in fasudil treatment. Furthermore, fasudil significantly suppressed the augmented responsiveness to methacholine induced by OVA challenges. CONCLUSION: Oral administration of fasudil inhibits eosinophil recruitment, goblet cell hyperplasia and airway hyper-reactivity by allergen challenges. These effects of this agent may be mediated by suppressing a chemokine and cytokines related to the pathophysiology of bronchial asthma such as eotaxin, IL-5, and IL-13. Our findings provide evidence that inhibition of the Rho/Rho-kinase pathway may be beneficial for bronchial asthma.

Molecular cloning and heterologous expression of an alpha-adrenergic-like octopamine receptor from the silkworm Bombyx mori.

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

[A case report of successful transcatheter arterial embolization therapy for osseous metastasis of hepatocellular carcinoma].

A 74-year-old man was admitted to our hospital with a chief complaint of severe local pain of the hip joint. Radiological findings showed a metastasized lesion on the left side of the pelvic wall originated from hepatocellular carcinoma (HCC) in the anterior segment of the liver. Transcatheter arterial embolization (TAE) therapy using epirubicin, Lipiodol and Spongel was successfully performed twice for primary HCC, and four times for osseous metastasis of HCC. After TAE therapy, the size of the metastasized lesion decreased with relief of pain, and an improvement in performance status of 4 to 2 was achieved. In conclusion, TAE therapy is thought to be very useful in the treatment of osseous metastasis of HCC with severe local pain.

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells.

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.

Intrahepatic interleukin-2 with chemotherapy for unresectable liver metastases: a randomized multicenter trial.

BACKGROUND/AIMS: A pilot study of Interleukin-2 (IL-2) with chemotherapy for unresectable colorectal liver metastases revealed a favorable response rate (76%). This prospective, randomized, multicenter study was conducted to evaluate the efficacy of this treatment protocol. METHODOLOGY: Over a period of 32 months, 46 patients with unresectable liver metastases were randomly assigned to 1 of 3 treatment groups: group A: chemotherapy alone, group B: chemotherapy plus high-dose, intermittent IL-2 (2.1 x 10(6) U twice weekly) or group C: chemotherapy plus low-dose, continuous IL-2 (7 x 10(5) U daily). Treatment continued for 4 weeks in the hospital and on an outpatient basis according to the clinical response. No crossover between treatment arms was permitted. RESULTS: IL-2 combined with chemotherapy produced a higher complete and partial response rate of 40% in group A, 60% in group B, and 78% in group C. Toxicity related to IL-2 included fever, chills, malaise, and eosinophilia. CONCLUSIONS: Hepatic arterial infusion of chemotherapy plus IL-2 resulted in an increased tumor response when compared with chemotherapy alone. To confirm the efficacy of this treatment protocol, we have started a large-scale, randomized, multi-institution trial.

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation.

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t1/2)five months) and were unstable in solutions of methanol and ethanol (t1/2

Decreased bone mineral density associated with early menopause progresses for at least ten years: cross-sectional comparisons between early and normal menopausal women.

To establish whether early onset of menopause carries an increased risk of osteoporosis, we compared the bone mineral density (BMD) of the second to fourth lumbar vertebrae (L2-4) between 18 women who had menopause before 43 years of age (early menopause group) and 19 women who had menopause after reaching 43 years of age (normal menopause group). Serum levels of calcium, phosphorus, calcitonin, intact parathyroid hormone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and alkaline phosphatase activity were measured, and urine samples were analyzed to derive calcium/creatinine, hydroxyproline/creatinine, pyridinoline/creatinine, and deoxypyridinoline/creatinine (D-Pyr/Cr) ratios. Mean BMD was significantly lower in the early menopause group than in the normal menopause group, and individual BMD values in about half of the subjects in the former group were below the fracture threshold for Japanese women. Serum concentrations of LH, FSH, and E2 were slightly, but not significantly, lower in the early menopause group than in the normal menopause group. The D-Pyr/Cr ratio was significantly higher in the early menopause group than in the normal menopause group. There was no correlation between L2-4 BMD and age or the number of years after menopause in the normal menopause group, but both age and the number of years after menopause were negatively correlated with L2-4 BMD in the early menopause group. These results indicate that BMD in women who have early menopause continues to decline for up to 10 years, and that menopause and aging increase the risk of osteoporosis.

Cytotoxicity of NaCl, a stomach tumor promoter, and prevention by rice extract in stomach mucosa of F344 rats.

Cytotoxicity of NaCl and its prevention by rice extract were studied in the pyloric mucosa of male F344 rat stomach after oral administration of rice extract and 2.6 M NaCl. Effect were observed histologically by hematoxylin and eosin staining and the bromodeoxyuridine method. Replicative DNA synthesis (RDS) was assayed by liquid scintillation counter with [3H]thymidine. NaCl (2.6 M) induced destruction of the surface mucous cells within 1 min. RDS and S-phase cells increased significantly (p < 0.01) and to a maximum at 17 h, and returned to control levels 48 h after exposure. Administration of aqueous rice extract 3 h before NaCl exposure reduced the morphological damage to the mucosa and prevented the increase in RDS dose dependently by up to 65% (p < 0.01). These results showed that NaCl induced rapid mucosal damage and cell proliferation in rat stomach mucosa and that rice extract prevented the damage and reduced the increase in RDS.

Cloning, subcellular localization and expression of CHL1, a subunit of magnesium-chelatase in soybean.

Mg-insertion is the first committed step in chlorophyll synthesis and is catalyzed by Mg-chelatase. In photosynthetic bacteria, bchI gene product was suggested to be a subunit of Mg-chelatase. We isolated a bchI homolog from a soybean cDNA library and designated it as chlI. CHLI consisted of 421 amino acid residues and the sequence exhibited a high similarity to other BchI homologs. CHLI contained an ATP-binding motif found in other BchI homologs. CHLI was localized in the soluble fraction in soybean chloroplasts, suggesting that it was a stromal subunit of Mg-chelatase. chlI mRNA in cell culture (SB-P) of soybean was reversibly induced by light.

A novel lipoxygenase from rice. Primary structure and specific expression upon incompatible infection with rice blast fungus.

A novel lipoxygenase cDNA (3,007 base pairs) was isolated from rice leaves (Oryza sativa cv. Aichiasahi) which had been infected with an incompatible race of the rice blast fungus, Magnaporthe grisea. A single copy of the gene is present in the rice genome and encodes a protein of 923 residues with a molecular weight of 102,714. This gene product shares the least amino acid sequence homology among plant lipoxygenases identified to date. A novel feature of this gene product is a putative transit peptide sequence at the amino terminus, suggesting the enzyme is localized in chloroplasts. An active lipoxygenase was expressed from the cDNA in Escherichia coli and characterized. The lipoxygenase introduces molecular oxygen exclusively into the C-13 position of linoleic and linolenic acids. The gene is expressed at high levels 15 h after inoculation with an incompatible race of M. grisea, at a low level after inoculation with a compatible race of the pathogen, and is not expressed in mock-infected leaves. Gene expression begins at the same time that the pathogen begins to penetrate into leaf tissue. This novel lipoxygenase gene expression is a part of the early response of the host to pathogenic attack.

Paeoniflorin improves learning deficit in 4-arm baited radial maze performance in rats with unilateral nucleus basalis magnocellularis lesion.

Paeoniflorin, a major constituent of peony root, has been demonstrated to attenuate scopola-mine-induced memory deficit in radial maze task in rats. To further investigate the therapeutic potential of this agent, the effects on a spatial learning deficit produced by unilateral lesion of the nucleus basalis magnocellularis (nBM) were examined using a 4-arm baited radial maze task in rats. Both working and reference memories were impaired by the nBM lesion. Daily treatment with paeoniflorin (0.1 and 1 mg/kg, PO) significantly improved the nBM lesion-induced learning deficits in terms of both working and reference memories. Paeoniflorin, however, did not attenuate the cortical choline acetyltransferase activity decreased by the nBM lesion. These data clearly demonstrate the beneficial effects of paeoniflorin on spatial cognition in rats.

Peony and its major constituent, paeoniflorin, improve radial maze performance impaired by scopolamine in rats.

A traditional Chinese medicine, Shimotsu-to has been shown to improve spatial working memory in rats. Shimotsu-to consists of four herbs, Japanese angelica root, cnidium rhizome, peony root, and rehmannia root. In the present study, the effects of aqueous extracts of each component herb on scopolamine (0.3 mg/kg)-induced spatial working memory disruption were examined using an eight-arm radical maze task in rats. Among the four component herbs, peony root extract (0.25 and 1 g dried herb/kg, PO) exhibited the most potent antagonizing effect on the scopolamine disruption of the choice accuracy. Japanese angelica root extract (1 g dried herb/kg, PO) also significantly attenuated the scopolamine disruption, whereas neither cnidium rhizome nor rehmannia root affected it. Paeoniflorin (0.01-1 mg/kg, PO), a major constituent of peony root, dose-dependently attenuated the scopolamine-induced impairment in the choice accuracy. Scopolamine (0.3 mg/kg, IP) significantly decreased the acetylcholine contents in the hippocampus, cortex, and striatum. Although paeoniflorin alone did not affect the acetylcholine contents, pretreatment with paeoniflorin significantly prevented the scopolamine-induced decrease in the acetylcholine content in the striatum, but not in the hippocampus or cortex. These data suggest that peony root mainly contributes to the cognitive enhancing effect of Shimotsu-to and that paeoniflorin may be one of the active constituents of peony root.

Antitumor effect of thermosensitive CDDP-liposomes on human osteosarcoma cells in culture.

We have administered cis-diamminedichloroplatinum (II) (CDDP) containing thermosensitive liposome and simultaneous hyperthermia into human osteosarcoma cells (OST) and studied the antitumor effects. Experiments were conducted under the following three different culture conditions as follows, (1) drug treatment alone, (2) hyperthermia after drug treatment, and (3) simultaneous hyperthermia and drug treatment. Antitumor effects were estimated by inhibition of DNA synthesis and decrease in cell growth rates. The antitumor action of simultaneous hyperthermia and drug treatments was the most dominant and synergistic among three experimental conditions. Accumulation of cellular platinum from thermosensitive CDDP-liposomes was markedly enhanced by hyperthermia. Flow cytometric analysis of the cell cycle indicated that CDDP-liposomes only under the simultaneous application of hyperthermia released free CDDP, and evoked cell accumulation in S and G 2/M phase. The results suggested that thermosensitive CDDP-liposomes were not taken up into cells by endocytosis, and released free CDDP into culture media under hyperthermia at 42 degrees C. From these, we have concluded that administration of thermosensitive CDDP-liposomes and simultaneous local hyperthermia could be a promising method for the treatment of osteosarcoma especially in the extremities.

A kampo prescription, shimotsu-to, improves scopolamine-induced spatial cognitive deficits in rats.

The effect of a Kampo (traditional Chinese medicine) prescription, Shimotsu-to, on spatial cognitive deficits produced by scopolamine was examined using an eight-arm radial maze and a T-maze. Scopolamine (SCOP; 0.075-0.3 mg/kg, ip) dose-dependently disrupted the radial maze performance. Single doses of Shimotsu-to (0.5 and 1.0 g/kg, po) as well as physostigmine (0.15 and 0.3 mg/kg, ip) improved the SCOP (0.3 mg/kg)-induced performance deficits in a dose-dependent manner. Shimotsu-to administered for 1 week in drinking water (0.5 and 1.0 g/kg/day) also exhibited dose-dependent reversal of SCOP-induced impairments in the radial maze performance. The same treatment improved SCOP (0.2 mg/kg)-induced impairments in T-maze delayed alternation performance. These data clearly demonstrated the beneficial effects of Shimotsu-to on spatial cognition.

[Influences of menopause and oophorectomy on bone metabolism and spinal bone mineral content].

The present study was performed on bone metabolism and spinal bone mineral content (BMC) in 3 groups of age- and body size-matched subjects: Thirty-three postmenopausal subjects, 37 oophorectomized (OPX) subjects and 22 premenopausal subjects. Serum levels of Alp and osteocalcin (OC) as indices of bone formation, U-Ca/cr and U-hydroxyproline (Hpr)/cr as indices of bone resorption, and Ca-regulating hormones, M-PTH, calcitonin (CT) and 1,25(OH)2D; were measured and spinal BMC was determined by QCT. Alp and OC levels were slightly higher in the postmenopausal group than in the OPX group and, in contrast, U-Ca/cr and U-Hpr/cr were slightly higher in the latter. The M-PTH level was slightly higher in the latter, and the CT level in the former. There was no difference in the 1,25(OH)2D level between two groups. For BMC, there was no difference between the two groups. The above results corresponded to the previously reported significant reductions in sex steroids. OPX seemed to affect bone metabolism more than menopause, there was no specific influence of OPX on spinal BMC as compared with QCT findings in postmenopausal subjects. Our previous study and the present study demonstrated that, at an interval of about 3 years after menopause or OPX, the endocrine system and bone metabolism tended to be more affected by OPX. However, BMC did not reflect any influence of OPX, or of menopause. There was no clear clinical difference between postmenopausal subjects and the OPX subjects with regard to the osteoporotic condition.

Effect of Panax ginseng on age-related changes in the spontaneous motor activity and dopaminergic nervous system in the rat.

Effects of Panax ginseng on the spontaneous motor activity and central dopaminergic systems in old rats were investigated and compared with those in young rats. Oral intake of a 1.8% water extract of Panax ginseng for four weeks produced an increase in spontaneous motor activity during the dark period in old rats, while it caused a decrease in the activity in young rats. After the intake of ginseng extract for five weeks, it caused a significantly low dopamine utilization in the daytime in the striatum of old rats, while it produced a high dopamine utilization in the structure of young rats. Concentrations of striatal dopamine D-2 receptors in old rats were significantly lower than that in young rats, although subchronic Panax ginseng did not affect the striatal D-1 and D-2 receptors of old rats. These results suggest that subchronic intake of ginseng extract inhibits the activity of nigro-striatal dopamine neurons in the daytime and activates spontaneous motor activity during the dark period in old rats, while it produces opposite effects in young rats.